I want to identify, separate and quantify S. aureus and P. aeruginosa from dual-species cultures both from planktonic or biofilm. Could there be any other method to do so without using flow cytometry or fluorescence proteins?
Thank you in advance,
Legesse
The most simple method would be culture, although you need to find a way to release the bugs from your biofilms in a quantitative way (e.g. sonication?). You could than use CNA agar medium (only the Staph will grow) and MacConkey agar (only the Psae will grow). If you work with defined cultures (i.e. only S. aureus and P. aeruginosa in your mixed culture), that should work. Even you inoculate a columbia agar plate, which supports growth of both bugs, you should be able to discriminate between both, since the growth characteristics are completely different.
In order to overcome the problems associated with aggregation and the resulting bias when a culture method is used you also can consider a PCR method.
Hi,
your question is from my "simple" times (second half of the last century):
1) DAPI is recommended for total counts. Medicine doctors sometimes prefer AO (me not)
2) Gram-staining is sufficient to quantify the proportion of bacteria.
3) Penicillin vs. Streptomycin or other antagonistic antibiotics are able to purify your cultures from a respective competitor (being G+ and G-)
Cheers
Mirek Macek
These are 2 different organisms with totally different characteristics. May be you increase the salt concentration to 7.5% in the medium where most of the P.aeruginosa will be inhibited and you can enumerate the Staph. aureus. In the other case use a common disinfectant like cetrimide in the medium and Staph will be inhibited and you can enumerate P. aeruginosa.
you can use differential enumeration. In a method which I am about to publish, i use nutrient agar containing 50 ug/ml chloramphenicol in the differential enumeration of Pseudomonas aeruginosa in river water samples. This antibiotic instigates P. aeruginosa to produce its characteristic greenish pigments. counting the pigment producing colonies should enable you quantify P. aeruginosa in the mixed culture. Also, the concentration of the antibiotic used is sublethal and will allow for the growth of many bacteria in a mixed culture. However, you should dilute your mixed culture to a dilution that will give your colony counts of hundreds on ordinary plate count agars. - a trick in my method. Good luck.
Thank you so much Lekieh Peekate. I am eager to read your work in this regard.
Have a nice time,
Legesse
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